| Objective:Gastric cancer is a common malignant tumor that seriously endangers human health,and the number of new cases and deaths of gastric cancer is increasing year by year as the problem of aging population in China becomes more and more serious.Therefore,it is urgent to investigate the mechanism of the development of gastric cancer and to achieve effective treatment of gastric cancer.It has been shown that the underlying cause of cancer susceptibility to chemoresistance and recurrence is the presence of a cell type known as cancer stem cells.Cancer stem cells are able to divide asymmetrically: on the one hand,they can give rise to daughter cancer stem cells,and on the other hand,they can differentiate to give rise to non-stem cancer cells(i.e.,common cancer cells that make up the majority of the tumor tissue).It is thus clear that cancer stem cells are the basis of malignancy heterogeneity.Cancer stem cells have several stemness characteristics,including high expression of drug efflux transport pumps,over-activation of antiapoptotic signaling pathways,ultra-high efficiency of DNA repair,latent characteristics of dormancy or slow proliferation,and accelerated metabolic patterns.It has been found that conventional cancer therapies(e.g.conventional radiotherapy)can lead to an increase in the proportion of cancer stem cells in cancerous tissues,implying that conventional cancer therapies are only able to kill normal cancer cells in cancer foci,but not cancer stem cells that are highly viable and able to migrate distally.Current targeted therapies for cancer stem cells are not ideal.Based on the identified properties of cancer stem cells,a series of novel therapeutic options have been designed,including targeting specific markers of cancer stem cells,molecular signaling pathways or stem cell niches and induction of apoptosis and differentiation of cancer stem cells.Drugs capable of targeting cancer stem cells have also emerged.Most of the therapeutic options have been tested at the laboratory stage to eradicate cancer cells.However,research statistics to date show that these new approaches are not effective in treating cancer.In addition,studies have shown that cancer stem cell targeted drugs have similar limitations as conventional anticancer drugs,such as off-target and unsatisfactory in vivo pharmacokinetics(poor stability,short duration of activity,poor distribution in vivo),and mediocre therapeutic effects.Since cancer stem cells are the key to cancer development,invasion and metastasis,resistance to radiotherapy and recurrence,and also the biggest obstacle to cancer treatment,it is necessary to reveal the characteristics of cancer stem cells and design novel therapeutic tools to specifically target cancer stem cells in order to effectively treat cancer and reduce the mortality rate of patients.Succinylation is a recently discovered post-translational acylation modification of proteins,which refers to the attachment of a succinyl group containing four carbon atoms to the lysine residue of a protein.The larger spatial structure of the succinyl group compared to the acetyl group,which contains only 2 carbon atoms,also means that succinylation is able to change the protein structure more strongly.Also,at physiological p H(p H 7.4),succinylation can change the charge state of protein lysine residues from +1 to-1,while acetylation can only change it from +1 to 0.Succinylation results in a greater charge change than acetylation modification,comparable to phosphorylation(from 0 to-2),and succinylation can bring about greater changes in protein structure and physicochemical properties than acetylation.Compared to acetylation,succinylation is able to bring about greater changes in the structure and physicochemical properties of proteins,and thus may have a more powerful function in regulating proteins.Referring to the important role of acetylation in the regulation of various cellular physiological activities including cancer stem cells,it is assumed that succinylation should also have similar regulatory functions.However,to date,no studies on succinylation modification in cancer stem cells have been reported.Therefore,this thesis investigates the identification of specific succinylation-modified protein marker molecules in gastric cancer stem cells using multi-disciplinary techniques such as proteomics,metabolomics,bioinformatics and cell biology,and elaborates their important roles in the development of gastric cancer,providing new theories for the stemness mechanism of gastric cancer and new ideas for the development of therapeutic drugs targeting gastric cancer stem cells.Method:1.Postoperative gastric cancer tissues and paracancerous normal tissue specimens were collected from gastric cancer patients of the First Affiliated Hospital of Anhui Medical University,Gastrointestinal(lumpectomy)Surgery Department,and lysine succinylation levels were analyzed by immunoblotting of proteins from gastric cancer tissues and paracancerous normal tissue specimens,respectively.2.Intraoperative tissue specimens from gastric cancer patients were analyzed by immunohistochemistry for lysine succinylation levels and histone methylation levels.3.Immunoblot analysis was performed to assess the effects of sodium succinate,resveratrol,and SIRT5 inhibitors on lysine succinylation levels in gastric cancer cells.4.CD44+CD54+ cells were isolated from gastric cancer cells MKN45 using magnetic bead sorting and self-renewal ability of CD44+CD54+ cells was detected by serum-free sphere-forming assay,and CD44+CD54+ cells were detected by real-time quantitative fluorescence PCR.CD54+cells and control cells using real-time quantitative fluorescence PCR to detect m RNA levels of the stemness marker Nanog and the differentiation marker KRT20.5.Succinylation proteomics assay to analyze the proteins undergoing lysine succinylation in CD44+CD54+ cells.6.Use of lentivirus to knock down the PHB1 gene in gastric cancer cells and construct a stable transgenic cell line.7.Use of lentivirus to construct a PHB1 succinylation 8.Metabolomic assays were performed to analyze the relative levels of metabolites in the stably transformed cell lines.9.The effects of sodium succinate,resveratrol,and SIRT5 inhibitors on self-renewal of gastric cancer cells were assessed using serum-free pellet formation assays.10.Flow cytometry was used to detect the gastric cancer cell surface markers CD44 and CD54.11.Real-time quantitative fluorescence PCR was used to detect Nanog and Kanog in stably transformed gastric cancer cells.m RNA levels of Nanog and KRT20 genes in stable gastric cancer cells.12.The effect of sodium succinate,resveratrol,and SIRT5 inhibitors on the tumorigenic ability of gastric cancer cells was examined using subcutaneous tumorigenesis in nude mice.13.The human primary tumor tissue human-derived tissue xenografts(PDX)model was used with sodium succinate and GSK-J4 intratumoral injection to detect the effect of drugs on graft growth.14.Detection of CD44,lysine succinylation and H3K27me3 localization and expression in human gastric cancer tissue specimens using immunofluorescence.15.Detection of α-ketoglutarate in human gastric cancer tissues and paired normal paracancer tissues using α-ketoglutarate(αKG)assay kit.16.Detection ofα-ketoglutarate in human gastric cancer tissue specimens and paired normal paracancer tissues using immunohistochemistry To detect the expression of H3K27me3 in human gastric cancer tissue specimens and paired paraneoplastic normal tissues.17.To analyze the expression of lysine succinylation in gastric cancer tissue microarrays using immunohistochemistry and to analyze the clinical prognosis relationship between lysine succinylation and gastric cancer patients.Results:1.The level of total lysine succinylation in human gastric cancer specimens was significantly lower than that in normal tissues adjacent to cancer(P<0.05).2.The level of H3K27me3 expression in human gastric cancer specimens was significantly lower than that in normal tissues adjacent to cancer(P<0.05).3.Treatment of gastric cancer cells with sodium succinate and SIRT5 inhibitors increased the level of total lysine succinylation and weakened the serum-free spherogenic ability of gastric cancer cells(P<0.05).4.CD44+CD54+ double-positive gastric cancer cells had lower succinylation level and stronger serum-free spheroplastic ability than normal control(P<0.05).5.CD54+ double-positive gastric cancer cells had higher m RNA levels of Nanog than normal control cells.m RNA levels of KRT20 in CD44+CD54+ double-positive gastric cancer cells were lower than normal control cells.6.Lysine at site 63 of PHB1 protein was desuccinylated in CD44+CD54+ double-positive gastric cancer stem cells,and its desuccinylation status significantly 7.The desuccinylation status of PHB1 significantly increased the α-ketoglutarate content in gastric cancer cells(P<0.05).8.Metabolomics suggested that the α-ketoglutarate content in the PHB1-k63 desuccinylated mutant(K63R)stable cells was higher than that in the PHB1-k63 succinylation mutant(K63E)stabletransformed cells and PHB1-k63 wild-type(WT)stable-transformed cells.9.Treatment of gastric cancer cells with sodium succinate,SIRT5 inhibitor and GSK-J4,respectively,inhibited their tumorigenic ability in nude mice,while resveratrol increased the tumorigenic ability of gastric cancer cells in vivo(P<0.05).10.Treatment of gastric cancer cells with sodium succinate and GSK-J4,respectively,decreased their Nanog expression levels and increased KRT20 expression levels(P< 0.05);whereas α-ketoglutarate and resveratrol treatment of gastric cancer cells increased Nanog expression and decreased KRT20 expression(P<0.05).11.Treatment of gastric cancer transplantation tumor mouse model with sodium succinate and GSK-J4,respectively,inhibited the growth of transplantation tumors(P<0.05).12.Lysine succinylation modification was lower in CD44 high expressing cells in human gastric cancer tissues(P<0.05).13.The expression level of H3K27me3 was lower in CD44 high expressing cells in human gastric cancer tissues(P<0.05).14.Cells with higher H3K27me3 expression in human gastric cancer tissues also had higher levels of lysine succinylation(P<0.05).15.α-Ketoglutarate was higher in human gastric cancer tissues than in paired normal paracancer tissues(P<0.05).16.H3K27me3 expression was higher in human gastric cancer tissues than in paired normal paracancer tissues(P<0.05).17.Immunohistochemical staining of human gastric cancer tissues revealed that lysine succinylation was generally lower in human gastric cancer tissues than in normal paracancer tissues,and lysine succinylation in hypofractionated gastric cancer tissues was lower than that in moderately and highly differentiated gastric cancer tissues.Gastric cancers with low succinylation were more likely to develop lymph node metastasis and had worse TNM stage,and lysine succinylation was associated with poor prognosis of gastric cancer patients.Conclusions:1.Total protein lysine succinylation levels were lower in human gastric cancer tissues than in normal tissues adjacent to the cancer,and total protein lysine succinylation levels were associated with poor patient prognosis.2.The present study revealed that the desuccinylated state of lysine at position 63 of PHB1 could promote the production of α-ketoglutarate in gastric cancer cells,which in turn could play a role in promoting the self-renewal of gastric cancer stem cells by decreasing the level of H3K27me3.3.Gastric cancer progression could be inhibited by increasing lysine succinylation and H3K27me3 methylation in gastric cancer tumors. |
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